HIV-1 must travel through the cytoplasm to reach the nuclear envelope (NE) of an infected cell, transport through a nuclear pore to enter the nucleus, and integrate its genome into the chromosomal DNA of the host cell. During this process, HIV-1 must reverse transcribe its RNA genome into double-stranded DNA, protect its genome and proteins from host degradative enzymes, and avoid triggering host defenses that would inhibit its replication. These postentry events are difficult to study biochemically because there are very few viral complexes in each cell; in addition, it has been difficult to study these events microscopically because methods to efficiently label and track viral complexes have not been available. _____Previous studies by other groups tracked fluorescently labeled (GFP-Vpr) HIV-1 complexes in infected cells and concluded that directional movements along microtubules (MTs) are required to reach the NE. However, infected cells contain viral complexes that have been taken up in endosomes as well as viral complexes that are released in the cytoplasm after fusion. Previous studies did not distinguish between fused viral complexes that can complete viral replication and viral complexes trapped in endosomes that cannot replicate. These studies may have observed MT-associated transport of viral complexes in endosomes, not fused viral complexes; thus, the functional relevance of directional cytoplasmic movements of viral complexes remains unclear. _____After fusion, the complex of HIV-1 viral capsid core, reverse transcriptase, and RNA carry out reverse transcription. The viral core is presumably dissociated from the reverse-transcribing complex in an ill-defined process called uncoating. Uncoating is seemingly required for nuclear import of the viral preintegration complex (PIC), since the HIV-1 core is too large for transport through a nuclear pore. Hulme et al. reported that the viral core becomes resistant to inhibition by TRIM5alpha, a restriction factor that binds to the viral core; this resistance to TRIM5alpha-mediated inhibition was interpreted as uncoating (PNAS 108:9975-9980, 2011). However, it is not clear whether the changes in the viral core that confer TRIM5alpha resistance are conformational changes that alter the structure of the viral core or involve loss of capsid protein (CA) from the viral capsid. Cyclophilin A (CypA) is a host factor that binds to the viral core; it is not known whether CypA binding affects the process of uncoating by stabilizing or destabilizing the viral core. Fluorescence microscopy assays are needed to determine the viral core integrity and to quantify the amount of CA associated with viral complexes. _____The HIV-1 PICs must associate with the NE before they can be imported into the nucleus. Nucleoporins (Nups) Nup153, Nup358, and TNPO3 have been reported to play important direct or indirect roles in NE docking and nuclear import. Arhel and colleagues observed docking of an HIV-1 complex with the NE in living cells but did not determine the functional relevance or the kinetics of these associations (Nat. Methods 3:817-824., 2006). We and others have observed association of HIV-1 complexes with the NE in fixed cells, but this approach cannot provide insights into the kinetics of HIV-1 association with the NE. We and others have also observed nuclear HIV-1 complexes in fixed cells. However, visualization of the transport of HIV-1 complexes from the NE to the nucleus has not been reported. Additionally, the movement of HIV-1 complexes in the nuclei has not been visualized. _____We recently observed that the APOBEC3F host factor tagged with yellow fluorescent protein (A3F-YFP) can be efficiently incorporated into HIV-1 virions and used the packaged A3F-YFP as a new tool to identify viral complexes in the nuclei of infected cells by an imaging approach. We will use A3F-YFP labeling of viral complexes to gain insights into the early stages of HIV-1 replication. Using thismethod, we showed that reverse transcription is not required for nuclear import of HIV-1 complexes, indicating that alterations in the viral CA structure that accompany reverse transcription are dispensable for their nuclear import. We found that HIV-1 CA mutations that altered the stability of the viral core significantly reduced the association of viral complexes with the NE and their nuclear import. In addition, we found that nuclear viral complexes remain close to the NE and are not randomly distributed in the nuclei. _____Our efforts in this project are focused on elucidating the structure and function of Vif and A3 proteins. The A3 proteins are among the most powerful host defense factors that, in the absence of Vif, completely suppress HIV-1 infection and spread. Therefore, determining the structure of the Vif:A3 interactions could provide opportunities for design of small molecules that can block this interaction and enable the A3 proteins to inhibit HIV-1 replication. We have noted that A3F remains stably associated with viral complexes, and have exploited this fact to develop a novel and robust system to study the behavior of HIV-1 complexes in infected cells. We are using this imaging approach to gain insights into the cytoplasmic movement, NE association, nuclear import, and nuclear movement of HIV-1 complexes. _____In our recent report on the dynamics and regulation of NE association and nuclear import of HIV-1 complexes, we note that these complexes exhibit an unusually long residence time at the NE (around 1.5 hours) compared to other cellular and viral cargos, and that HIV-1 CA and host Nup358 are required for NE docking and nuclear import. Soon after import, the viral complexes exhibit a brief fast phase of movement, followed by a long slow phase, during which their movement is similar to that of integrated proviruses, suggesting that they rapidly become tethered to chromatin through interactions that do not require LEDGF/p75. Importantly, we found that NE association and nuclear import is regulated by the CA-cyclophilin A interaction, but not reverse transcription, and that one of the viral core uncoating steps, characterized by substantial loss of CA, occurs concurrently with nuclear import. These studies provide valuable insights into the mechanisms by which HIV-1 gains entry into the nuclei of infected cells. Inhibition of the nuclear import of viral complexes could lead to development of novel anti-HIV drugs.